665
Development 103, 665-674 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Detection of spatially- and stage-specific proteins in extracts from single
embryos of the domesticated carrot
R. H. RACUSEN1 and F. M. SCfflAVONE
Plant Development Laboratory, Department of Botany, University of Maryland, College Park, MD 20742 USA
Summary
Single embryos, representing each of four distinct
morphological stages, were selected from cultures of
the domesticated carrot for analysis of total [3SS]methionine-labelled proteins. Following exposure to radiolabel for 12 to 18 h, embryos were individually disrupted in a 3 mm diameter, precisely-matched, plastic
mortar and pestle. Radiolabelled proteins extracted
by this procedure were separated by two-dimensional
electrophoresis procedures, consisting of isoelectric
focusing in 1 mm tubes, followed by SDS-PAGE in a
small slab gel. Comparisons of autoradiographs of
these gels revealed that the levels of a number of
proteins were modulated during the conversion of
disordered callus cells into maturing embryos. In
addition, miniature surgical techniques were used to
separate the apex (cotyledon end) from the base (root
end) of late-stage embryos, for extraction of proteins
and analysis of spatial differences in protein distribution. About five proteins in extracts from each
section were observed to be synthesized at different
rates in the two halves, indicating that there are
molecular correlates for early polarized growth.
About half of the proteins, whose appearances were
unique to apical and basal sections of embryos, were
also observed to fluctuate in comparisons of autoradiographs of two-dimensional protein separations from
embryos at different developmental stages.
Introduction
such mechanisms in development is lacking; however,
measurements of electric fields around carrot embryos (Brawley et al. 1984), which appear to be
aligned with the axis of embryo elongation, indicate
that nonmolecular modes of information transfer
clearly exist in these organisms.
An alternative explanation for the absence of a
sizeable number of stage-specific proteins in the
earlier studies is that the design of these experiments
may have militated against the detection of novel
polypeptides. For example, examinations of total
proteins contained in whole-organism extracts were
typically conducted as comparisons between undifferentiated cells and a mixed population of various
stages of embryos. Such an experimental design
presumes that callus cells will uniformly exhibit a
protein complement typical for disorganized growth
and that, once initiated, all embryos will display
proteins characteristic of ongoing differentiation. It
does not provide for the possibility that groups of
undifferentiated cells may synthesize overlapping
portions of the total protein complement necessary
In contrast to the conspicuous molecular changes that
accompany developmental transformations in other
well-characterized systems, such as Caenorhabditis
(Johnson & Hirsh, 1979; Sulston et al. 1983), Dictyostelium (Alton & Lodish, 1977; Barklis & Lodish,
1983) and sea urchin (Davidson et al. 1982; Brandhorst et al. 1983), the present literature on gene
expression in somatic embryos of carrot suggests that
perhaps only 1-2 % of the proteins detected on twodimensional electrophoretic gels appear or disappear
when one compares extracts of disorganized cellular
clumps (callus) and whole embryos (Sung & Okimoto, 1981, 1983). This paucity of detectable shifts in
temporal gene expression has fostered the notion that
the majority of genetic events that regulate carrot
embryogenesis may occur in advance of visible
changes in morphology; the changes in form being
hypothetically coordinated by physical or metabolic
factors that do not require new gene expression (Sung
et al. 1984). Evidence for the direct involvement of
Key words: somatic embryo, carrot, two-dimensional
electrophoresis.
666
R. H. Racusen and F. M. Schiavone
for ordered growth, nor is it possible to be certain that
embryos, which may have aborted in development,
have not reverted to the production of some callustype proteins. Further, the use of mixtures of embryos to prepare extracts precludes comparisons between the conventional embryo stages, which are
presently defined on the basis of anatomic features.
Some of these problems have been alleviated by
the extraction of proteins from embryos that have
been sorted by stage following successive passes
through graded sieves (Giuliano et al. 1983). This
refinement has permitted the characterization of a
specific antibiotic resistance (Pitto et al. 1985) and the
cloning of three cDNAs which are purportedly correlated with transitions between embryo stages (Choi et
al. 1987). The criteria for separating embryos in these
studies, however, continues to rely on rather rudimentary differences in embryo shape; thus, there is
no direct assurance that the mixture of organisms
sustains comparable homogeneity at the molecular
level. Considering the impressive lower limits of
detection which contemporary procedures in molecular biology are capable of delivering, even small,
unsuspected amounts of contaminating material
could produce misleading results.
In this paper, we report a reexamination of stagespecific differences in gene expression during carrot
embryo development by comparing two-dimensional
gel separations of total proteins extracted from single
embryos. To ensure that each embryo was accurately
categorized by morphological stage, we followed a
two-phase selection protocol. From subcultures of
embryos that had been sorted into size ranges by
passage through screens, we manually selected representatives according to a schedule of geometric
measurements that we found in earlier work (Schiavone & Cooke, 1985) and which provides more
precise demarcations between adjacent stages. Since
we also wished to explore the possibility that certain
genes might be expressed in a tissue-specific manner,
we analysed extracts of portions of single embryos
that had been removed by microsurgery.
Materials and methods
Carrot cell culture
Cells, originally derived from young hypocotyls of Daucus
carota L. cv. Danvers, were maintained in shaking 125 ml
suspension cultures at 25°C in dim room light. The medium, traditionally used for growing these cells in an
undifferentiated state (Murashige & Skoog, 1962), contained 5/jM-2,4-dichlorophenoxyacetic acid (2,4-D) and
was changed at weekly intervals. Embryo formation was
initiated by procedures detailed in Schiavone & Cooke
(1985), in which the cells were transferred to the same
medium without 2,4-D (MSE medium). Embryo cultures,
8-10 days old, were passed through first a 380pm sieve and
then a 117um sieve. In this procedure, heart- and torpedostage embryos are captured on the 117/an sieve, while
oblong- and globular-stage embryos and a small amount of
undifferentiated cells were rinsed through. Heart- and
torpedo-stage embryos were transferred to a dry Petri dish
and enough MSE added to dilute the embryos to approximately 50 embryos ml"1. From this dish, single embryos
were selected with a Pasteur pipette while being viewed
under a dissecting microscope at xlO.
Selection and transfer of individual embryos and
callus
To determine the stage of a particular embryo, we made
camera-lucida drawings of each embryo, tracing the embryo's periphery using methods and criteria developed by
Schiavone & Cooke (1985). Briefly, any embryo whose
outline also corresponded to a complete circle was termed a
globular-stage embryo. Embryos that had undergone polarization along the future root-shoot axis (thus having an
axial length greater than the embryo width), but still
maintaining a smooth apical end were considered to be in
the oblong stage. Heart-stage embryos, like the oblongstage, are clearly longer than wide, but are in the process of
cotyledon formation. These embryos have an apical end
which is not part of a circle, but contains two prominent
cotyledonary bulges at the apex. Since we were unable to
provide a clear morphological distinction between heartand torpedo-stage embryos in the earlier studies, we
denned torpedo-stage embryos as those cotyledon-bearing
embryos greater than 400/jm in axial length. Callus was
maintained in medium containing 2,4-D and two or three
clumps of these undifferentiated cells, ranging from 50 to
100/im were utilized for protein extracts. Embryos and
callus were transferred to 200/il of fresh MSE medium in
wells of an Elisa plate for use in the surgical and protein
extraction procedures that followed.
Application of radiolabel, surgery and tissue
extraction
A. 5Ou\ droplet of sterile medium was placed in a shallow
parafilm well, which had been formed by pressing a sterile
strip of this pliable material with a gloved index finger
against the 0-5 cm square openings of a 0-5 cm thick plastic
grid. With a 5x7cm section of the grid, it was possible to
form 16 of these shallow wells, such that each was separated
on all sides by unused cells. There were several advantages
in using these multiwell plates. First, the hydrophobic
nature of parafilm kept the droplet spherical so that
subsequent microlitre additions and withdrawals could be
performed with minimal wetting of the well or the transfer
tip. Second, the entire grid was nested in a covered plastic
tray containing a few ml of water, which slowed evaporation
of the droplets during labelling procedures. Third, the grid
could be placed under a dissecting microscope for removal
or addition of embryos to the droplets. Because the
droplets protruded above the sides of the well and tended to
remain centred in the depression, it was relatively easy to
manoeuvre the pipette tip in from any angle to retrieve a
particular embryo. Finally, the parafilm surface could be
Proteins in carrot embryos
simply stripped and discarded following the labelling procedure.
Individual embryos were sterilely transferred in 2/il of
medium with a microlitre pipetting device to the 50 y\
droplet in the shallow, parafilm wells. Generally 1/jl of L[35S]methionine (Amersham; 50-lTBqmmol"1), consisting
of 555 kBq total activity was added to 50^1 droplets,
containing 1-4 embryos. The tray was then covered, placed
in darkness and allowed to incubate for 18 h at 25 °C.
Labelled embryos were washed by nonsterile transfer in a
2fi\ volume to a fresh 50 jA droplet of culture medium in a
shallow well identical to the ones used for labelling. This
procedure was repeated two more times, with the final
transfer being made into an extraction buffer consisting of
25 mM-Tris-HCl, pH7-8; lOOmM-KCl; 0-5mM-MgCl2;
10 % Triton X-100; 200mM 2-mercaptoethanol and 0-lmMphenylmethylsulphonyl fluoride. The number of counts in
the final wash was typically less than 0-1% of the total
added.
To separate apical and basal sections for protein analysis,
embryos, viewed at xlO under a dissecting microscope,
were severed at the midpoint along the longitudinal axis,
using a tiny scalpel that was fabricated according to
methods from Lowry & Passonneau (1972). The cutting
edge of this instrument is a shard of a double-edged razor
blade about 0-5 mm in length, which was glued to the end of
0-5 cm long, single toothbrush bristle. The other end of the
bristle fibre was glued to a dissecting needle. For the cutting
operation, selected embryos were transferred with a Pasteur pipette in about 20^tl of medium to the bottom of a
sterile, 100mm Petri dish. We found that embryos could be
sectioned with a single, clean cut by first positioning the
blade edge parallel to the bottom surface of the plastic dish.
While maintaining the parallel orientation as closely as
possible, the blade was then positioned over an embryo at
the site where the cut was to be made, and the blade
brought straight down to separate the tissues.
Under a dissecting microscope at x25, individual washed
embryos or sections were transferred in a 2^1 volume to the
surface of a 3 mm diameter, conically shaped, teflon pestle.
The pestle with droplet was pushed into a tightly fitting,
1 cm long, polypropylene mortar, the opposite end of which
was formed to precisely mate with the conical pestle. The
tip of the conical end of the mortar possessed a 0-5 mm
opening. Using moderate finger pressure and two or three
rotations of the pestle, embryos were crushed between the
closely matched surfaces of this apparatus. The pestle was
then slowly withdrawn and 3-8 y\ of additional cold extraction buffer from a microlitre pipette were allowed to be
drawn in through the hole in the tip of the mortar. The
extract fluid of 5-10 jA was efficiently removed by lmin
centrifugation in a cold Eppendorf centrifuge; fewer than
0-1% of the total counts incorporated into embryos
remained in the components of the extracting device.
Extracts were sonicated for 2 min by placing the tips of the
Eppendorf tubes in a bath sonicator filled with ice water.
The tubes were again centrifuged for 1 min and the extracts
frozen or analysed immediately.
Two-dimensional gel electrophoresis
To determine total [35S]methionine uptake into embryos,
667
protein extracts were occasionally added to 5 ml of a
scintillation cocktail (Scintiverse II, Fisher Scientific) and
counted in a Beckman model LS 7000 scintillation counter.
Without disturbing the pellet in the Eppendorf tube, 3-8 /il
of most extracts were loaded onto 6 cm long, 1 mm diameter, isoelectric focusing gels. The polyacrylamide-based
gel mixture has been described previously (O'Farrell, 1975)
and was made with 2 % of 3/10, 4/6 and 5/7 ampholytes
(Bio-lyte; Biorad, Richmond, CA, USA). The gels were
prefocused at 120 V for 1 h and were run with extracts for 12
to 18 h at 120 V. Completed IEF runs were always followed
immediately by electrophoresis in the second dimension on
a 8cm wide x 6cm long x 0-75 mm thick, 2 % SDS, 10%
polyacrylamide slab gel (Laemmli, 1970). The pH gradient
formed in the focusing tube was determined by measuring
the pH of degassed solutions of 0-025 M-KCI, containing
equilibrated, 0-5cm sections of a focused gel. The relative
molecular masses of separated proteins were determined
from MW standards (Bio-rad) that were loaded in a
separate lane, adjacent to the well containing the firstdimension tube gel. Completed SDS-polyacrylamide slabs
were stained in Coomassie Blue solution, destained and
dehydrated between cellophane sheets in a commercial gel
drier. The dried gel was then sandwiched against Kodak
X-O MAT X-ray film in a standard cassette and exposed for
3 to 10 days at -80°C. Comparisons of autoradiographs
from two-dimensional gels of different embryo extractions
were done by side-by-side visual inspection. To standardize
this somewhat subjective procedure, we used a type of
'constellation analysis' in which we drew interconnecting
lines between suggestive groupings of spots on x3 photographic enlargements of the autoradiograph films. Since
there is a certain amount of variability in the degree of
separation in each dimension on the gels and in the amount
of radioactivity supplied from each extract (see results
section on incorporation, below), we registered only those
spots where a similar change in intensity occurred in three
separate gels of extracts from embryos and callus of the
same age and approximate size.
Results
Incorporation of radiolabel
Table 1 shows total amount of [35S]methionine taken
up by callus and embryos at different stages. In
general it appeared that there was a direct relationship between the size (measured as axial length or
total protein) of an embryo and the amount of label
incorporated. The transport of methionine was highly
dependent on the presence of a carbon source in the
medium; incubation of heart embryos in growth
medium lacking sucrose resulted in 95 % loss in
accumulation of the isotope (data not shown).
Two-dimensional gel electrophoresis of embryo
extracts
Analysis of the total sulphur-containing proteins by
this technique revealed, in the more heavily loaded
gels, about 200 spots, which are catalogued in Fig. 1.
668
R. H. Racusen and F. M. Schiavone
21-5
Fig. 1. Two-dimensional gel electrophoresis of [35S]methionine-labelled polypeptides from a single torpedo-stage carrot
embryo. The autoradiograph has been purposely overexposed to reveal proteins that were synthesized in lower
amounts. All the spots that were reproducibly seen to appear in the course of running many such gels with extracts of
embryos were numbered with a single digit-single letter designation, starting from the high molecular weight, basic
corner of the slab gel. Since certain spots, which are apparent in other embryos, are not visible in autographs of
torpedo-stage proteins, the ' + ' sign is used to indicate their positions.
Proteins in carrot embryos
669
Table 1. Total incorporation of [SJmethionine into callus cells and somatic embryos of the domesticated
carrot (means ± S.E.)
Somatic embryos
Axial length (jim)
Total protein (//g)
Total incorporation (cpm x 10*)
Callus
Globular
Oblong
Heart
Torpedo
NA
8-88 ± 103
2-3 ± 0-4
113 ± 5
0-83 ±006
0-3±01
178 ±7
1-86 ±0-29
1-5 ± 0 1
430 ±18
5-91 ±0-98
2-5 ±0-2
899 ±57
8-88 ± 1 00
3-6 ±0-2
About 50 of these proteins produced the most intense
spots, and we estimated that these represented about
90% of the protein extracted from the embryo, as
follows. First, we summed the areas of the darker
spots by tracing them on a tablet digitizer (Jandel
Scientific, Sausilito, CA, USA), coupled to a microcomputer. We then compared the optical density of
one of the darker spots with a lighter one, and using
Beer's law deduced that the darker regions were
produced from the conversion of about 10 times as
many silver grains in the film. Assuming that the
remaining 10% of silver grains, reduced by isotope
decay, were equally divided between the 150 lighter
spots would imply that individual spots detected on
this film comprised as little as 0-05 % of the total
protein extracted from the embryo. This is about 25
times higher than the apparent threshold for detection that has been experimentally determined (Johnson & Hirsh, 1979), which suggests that protein
analysis by these methods may be extended down to
the extremely small extraction volumes used in our
assays without unacceptable losses in resolution.
Fig. 2 shows an example of a two-dimensional gel
obtained with an extract of callus, containing about
5 /ig total protein as determined by the Lowry method
(Lowry et al. 1951). In this autoradiograph, three
spots are highlighted with arrows which, using the
catalogue in Fig. 1, correspond to numbers 4d, 6u and
7c. These proteins were produced at much lower
levels following the transition to early stage embryos,
but reappeared in certain later stage embryos. As
described below, synthesis of these polypeptides also
appeared to be restricted to basal regions of the later
stage embryos. Autoradiographs of two-dimensional
gels from single globular embryos were lighter in
appearance owing, most likely, to the tiny size and
lower radiolabel uptake of embryos at this stage
(Fig. 3A). As a consequence, we felt that the loss of a
particular spot from these autoradiographs would not
be a reliable indicator of a decline in synthesis of a
protein. The oblong-stage autoradiograph showed
the appearance of three polypeptides that were not as
actively synthesized in callus cells (Fig. 3B). These
are highlighted with arrows and correspond to numbers 2m, 3a and 6b. The appearance of a fourth
protein, which was incompletely resolved, is also
21-5-
Fig. 2. Two-dimensional autoradiograph of
undifferentiated callus cells, containing about 5^/g total
protein. The autoradiograph has been overexposed to
reveal proteins that were synthesized in lower amounts.
Three polypeptides, indicated with arrows, disappear in
subsequent autoradiographs of protein extracted from
embryos (see results).
indicated (number 2a). Three other proteins (arrows)
later declined in synthesis, either at the heart stage
(number 4v), or the torpedo stage (numbers 3a and
6b). In autoradiographs of heart stage gels, we noted
three proteins that exhibited an increase in intensity
over those seen in the oblong-stage films (Fig. 3C).
Two of these, which did not resolve into well-defined
spots (numbers 2a and 2i), subsequently decreased in
intensity in the torpedo stage. The appearance of the
other enhanced protein (number 4d) apparently
bracketed the oblong stage, being found additionally
in callus and the base-section extracts of torpedo
embryos. Two other proteins (numbers 6a and 6b)
670
R. H. Racusen and F. M. Schiavone
i
B
Fig. 3. Two-dimensional autoradiographs of extracts of individual embryos from each of the four recognizable stages of
embryo development: (A) globular, (B) oblong, (C) heart and (D) torpedo. Autoradiographs of globular-stage embryos
were typically as light as the one shown here, perhaps due to the lower rate of radiolabel incorporation and smaller size
of these embryos. Autoradiographs of separations of proteins from the other three stages are marked with arrows to
indicate those proteins whose levels increased or decreased between adjacent stages (see Results). Vertical and
horizontal tick marks correspond to approximate molecular weights and pH shown in Figs 1 and 2.
Proteins in carrot embryos
were seen to be synthesized at much lower rates
following the passage into the torpedo stage. In
addition to these declines in synthesis of certain
proteins, extracts of torpedo-stage embryos (Fig. 3D)
showed the return of two proteins that were last seen
in callus (number 6u) and oblong-stage embryos
(number 4v). A small amount of synthesis of a
protein (6w), not seen in earlier stages, also appeared
in torpedo-stage embryo extracts.
Gels from extracts of sectioned embryos
Differences in the spatial distribution of a number of
proteins were evident in autoradiographs of twodimensional gels from apical and base extracts of
torpedo-stage embryos. In surgically bisected embryos, three proteins were restricted to the half
containing the apical (cotyledonary) region (Fig. 4A;
numbers 4k, 7n and 7o) and six proteins were
synthesized in higher amounts in sections with the
suspensor (root/hypocotyl) pole (Fig. 4B; numbers
4d, 4v, 7a, 7c, 8q and 9m). Interestingly, certain of
these spatially distinct proteins were identical to those
whose rates of synthesis were modulated during
transitions between developmental stages. For instance, apical end protein, number 6a, first appeared
in heart-stage embryos. Similarly, base end protein
number 7c appeared in callus, but was synthesized in
671
lower amounts in all embryo stages until torpedo; and
protein number 4d, synthesized in callus, was absent
in radiolabelled form in extracts of embryos until the
heart stage. A summary of stage- and spatially
specific changes in protein synthesis are shown diagrammatically in Fig. 5.
Discussion
Developmental processes such as plant embryogenesis are undoubtedly coordinated by transfer of
information between different cells or between compartments in individual cells. The passage of characteristic form between parent and offspring as a
heritable trait implies that the ultimate store of
instructions resides in the genome, but it is not certain
if the successive expression of particular genes is the
mechanism whereby the temporal framework for a
developmental transition is established. In certain
systems, for example, there is evidence that cellularly
derived electrical fields (reviewed in Jaffe & Nuccitelli, 1977), diffusing chemical 'morphogens'
(reviewed in Meinhardt, 1982) or physical stresses in
a cellular matrix (Lintilhac, 1984) may be the primary
effecters of observed changes in shape.
In interpreting these, or other experiments, utilizing sensitive techniques in protein or nucleic acid
B
•
* ~
Fig. 4. Two-dimensional autoradiographs of extracts from an apex (A) and a base (B) of a surgically bisected torpedostage embryo. Arrows indicate polypeptides that were unique to the arrays of proteins from each tissue section. Vertical
and horizontal tick marks correspond to approximate molecular weights and pH shown in Figs 1 and 2.
672
R. H. Racusen and F. M. Schiavone
IEF-
7
xicr3
C,G,O. H
92-5-
o
66-2
5k
45C,G,O,H,Ta
H
8o
O
21-5
Fig. 5. Diagrammatic summary of the changes in the levels of synthesis of polypeptides extracted from carrot callus and
embryo tissues. This tracing of an autoradiograph of a two-dimensional electrophoretic separation of proteins from
carrot cell extracts includes many of the more intense spots, but omits the lighter ones. All spots are numbered
according to the system described in Fig. 1. The protein spots that were observed to change during embryo
development, or were found only in apex or base sections of embryos, are indicated by lines connecting them to letter
designations of the stages, or positions, in which they were present. In this scheme, C, callus; G, globular; O, oblong;
H, heart, T, torpedo; T a , apex of torpedo; T b , base of torpedo.
analyses to perform broad molecular comparisons
between organisms which differ in outward appearance, one must take care not to overextend the
postulates of gene activation, which were originally
put forth to explain the directional biasing of metabolism through the synthesis of key enzymes. Therefore, we issue the following caveats in advance of
considering these findings. First, the detection of
proteins by the methods used in this investigation is
limited to sulphur-containing proteins with isoelectric
points between 4-5 and 7-5. Second, the differences in
intensities of spots on autoradiographs of electrophoregrams only provide information about the relative abundance of proteins synthesized during the
application of label, and furnish no direct measure of
the total abundance of any protein in the tissue
extracts. Third, the search for stage- or tissue-specific
changes in polypeptide composition, following incorporation of radiolabel, is prejudiced to identify only
those events that are accompanied by appearance of
new proteins; the possibility that a developmental
transition might be cued by the degradation of an
Proteins in carrot embryos
existing polypeptide is not accommodated in the
experimental design. Finally, the identity and function of proteins detected in two-dimensional gels are
unknown, as is their role, if any, in promoting the
progression of morphogenesis.
The changes in the appearance of proteins in our
examination of extracts of single embryos fall into
three general classes: (1) proteins that were observed
in one or two stages of embryos, (2) proteins that
were observed in callus cells and certain embryo
stages and (3) proteins that were apparently localized
to apical or basal portions of a sectioned embryo. At
the level of detection in these experiments, we did not
identify any proteins that were unique to extract of
callus cells. Of the 15 proteins that were determined
to be either stage- or tissue-specific, 9 were present in
callus. Interestingly, 3 of these 9 proteins were not
found in autoradiographs of early embryo stages
(globular, oblong and, in one case, heart) but reappeared in extracts of basal portions of torpedostage embryos. These observations raise the possibility that the expression of certain genes might occur
in a polarized fashion, at least in later stages of
embryo development. Whether they are similarly
expressed with respect to position in earlier stages, or
perhaps in regions of callus that are to become the
suspensor end of the embryo, are intriguing questions
that may ultimately be approached by analysis of
extracts from surgically removed tissues from
younger organisms.
Nine of the radiolabelled proteins that were found
in extracts of various stages of embryos were also
determined to be asymmetrically distributed into
apical or basal halves of sectioned torpedo embryos.
Since the establishment of polarity is the pivotal
morphological event which signals the conversion to
organized growth, it is tempting to consider the
possibility that early, spatially polarized gene expression gave rise to these protein distributions,
which then might serve as molecular determinants of
the ensuing polarized morphology. It is equally possible, of course, that these protein differences simply
represent fundamental biochemical differences between the cell types in the apical and base regions.
Again, analyses of extracts from earlier stage embryos that have been surgically sectioned would be
necessary to determine the onset of such differences,
in turn reinforcing or repudiating the notion that they
have causal significance.
The most provocative changes in newly synthesized
polypeptides that we observed were ones in which a
protein appeared in one stage, was not detected in
one or two following stages and then reappeared in an
even later stage. There were four examples of such
episodic synthesis in our survey, three of which began
in the callus cells. Assuming it can be shown that
673
these gaps in the synthesis of proteins are useful
preindicators of specific embryogenic transitions, it
will be important in future experiments to determine
what happens to the level of these polypeptides when
they are not being synthesized. If they are not
degraded during the stages when they are absent from
autoradiographs, then each ensuing period of synthesis would raise the total level, creating a step-wise
accumulation of the polypeptide as embryos
matured. If, on the other hand, the protein is
degraded in the intervening stages of low synthesis,
then an oscillation in the level of the peptide would
occur. Either of these two possibilities could, in
theory, serve as an effective means of signalling a shift
in the activities of cells in the embryo.
Whatever may be their role in development, it is
clear that the modulation in the levels of synthesis of a
number of polypeptides in carrot embryos is more
complex than has been previously appreciated. Comparisons between extracts of callus and extracts of a
mixture of embryos, as has been done previously,
would not have permitted about 50 % of the stagespecific differences to be detected, leading us to
confirm the earlier conclusions that carrot embryogenesis is accomplished with the addition of only a
few new gene products. Microsurgery of individual
embryos proved to be a useful addendum to the single
embryo extraction procedures; the combined methodologies provide a means for directly identifying
proteins that are spatially segregated into different
regions of a developing embryo.
The authors wish to thank Drs Gary R. Pasternack and
Frank P. Kuhajda of the Johns Hopkins University, School
of Medicine, for the use of their laboratories and for
guidance in some of the techniques.
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(Accepted 11 April 1988)